Insect Trichoplusia ni High Five (Hi-5) cells were cultured in an incubator at 27°C using Grace’s Insect Medium with 10% fetal bovine serum (WISENT, Nanjing, China) following the manufacturer’s instructions. Analogously, S. frugiperda 9 (Sf9) cells were cultured using Sf-900™ II SFM and D. melanogaster Schneider 2 (S2) cells were cultured using Schneider’s Drosophila Medium at 27°C. Furthermore, human embryonic kidney 293T (HEK 293T) cells were grown in Dulbecco’s modified Eagle’s medium at 37°C and 5% CO₂ containing 10% FBS and penicillin‒streptomycin. These cultured media were purchased from Life Technologies Corporation.

Raw data on the genome, transcript, and protein sequences were retrieved from the NCBI and InsectBase 2.0 (Mei et al., 2022). To identify homologous genes in different species, tBLASTn analysis was performed using the Trichoplusia ni chloride intracellular channel (TnCLIC) protein sequence as a query (e-value < 1e-5). Extracted contigs were predicted using the SoftBerry FGENESH program (Solovyev et al., 2006) and manually checked by comparing them in the GenBank database. The SignalP 5.0 server was used for signal peptide prediction (Almagro Armenteros et al., 2019). Collected homologous protein sequences from 110 insect species were aligned using MAFFT (Katoh and Standley, 2013) and trimmed using TrimAL (Capella-Gutierrez et al., 2009). Then, maximum-likelihood phylogeny was calculated using IQ-TREE (version 2.0.3) (Nguyen et al., 2015) under the LG + G4 substitution model with 1, 000 ultrafast bootstraps. The phylogenetic tree was then optimized and visualized via iTOL (Letunic and Bork, 2021). For multiple sequence alignment, protein sequences of TnCLIC, SfCLIC, DmCLIC, NlCLIC, PpCLIC, TcCLIC, Exc-4, and human CLIC4 were aligned using ggmsa R package (Zhou et al., 2022) and the consensus sequence logo was generated to show the frequency of amino acids (https://github.com/YuLab-SMU/supplemental-ggmsa).

Total RNA was extracted from cells using TRIzol reagent (Invitrogen, CA, United States) according to the manufacturer’s protocol. Then, RNAs were reverse-transcribed into single-stranded complementary DNAs (cDNAs) using the TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). Cloned PCR amplifications with restriction site sequences (KpnI and XbaI) of TnCLIC were carried out using KOD One™ PCR Master Mix (TOYOBO, Shanghai, China). Primers were designed by CE Design (Vazyme, Nanjing, China) and are listed in Supplementary Table S1. Afterward, pCold-I plasmids digested by FastDigest KpnI and XbaI (Thermo Fisher, CA, United States) were recombined with PCR products using the ClonExpress Ultra One Step Cloning Kit (Vazyme, Nanjing, China). After sequencing, the recombinant plasmid was transformed into BL21 Chemically Competent Cell (TransGen, Beijing, China).

To induce protein expression, 1 mM isopropyl-β-d-thiogalactoside (IPTG) was added when the OD value of the bacterial culture (grown at 37°C) reached 0.5–0.6. After 24 h of incubation with 120 rpm shaking at 15°C, the recombinant E. coli cells were harvested by centrifugation and lysed with BugBuster® Master Mix (Novagen, MA, United States) at 4°C. Since the pCold-I vector is equipped with a 6 × His tag, recombinant proteins were purified by His-tag affinity chromatography using cOmplete His-Tag Purification Resin and Ni-NTA BUFFER KIT (Merck, United States) according to the manufacturer’s instructions. The purified proteins were then desalted using Zeba™ Spin Desalting Columns, 7K MWCO, 2 mL (Thermo Scientific, Waltham, United States). SDS‒PAGE and western blot analyses were performed after the protein concentration was determined using Quick Start Bradford Dye Reagent (Bio-Rad, CA, United States) (Bradford, 1976). The mouse monoclonal antibody (a label-free primary antibody) against 6 × His tag was purchased from GenScript (Nanjing, China). Finally, the TnCLIC antibody was a rabbit polyclonal antibody made by HuaAn Biotechnology Co., Ltd. (Hangzhou, China) using the purified protein.

Before siRNA knockdown experiments, cells were plated and grown until 60%–80% confluence. The next day, cells were transfected with siRNA (100 nM final concentration) using Lipofectamine™ RNAiMAX Reagent (Invitrogen, CA, United States) in OptiMEM medium (Life Technologies Corporation, NY, United States) following the manufacturer’s instructions. Two pairs of siRNA per gene were designed and synthesized by Sangon Biotech (Shanghai, China). The siRNA sequences from 5′ to 3′ were as follows: siRNA-TnCLIC-1 and siRNA-SfCLIC-1: GCA​AGU​ACU​UCG​UCG​ACU​UTT; siRNA-TnCLIC-2: GCC​UCG​CAA​UAC​UGG​AGA​ATT; siRNA-SfCLIC-2: GCA​GAG​GGA​CUA​GAU​UCU​UTT; siRNA-DmCLIC-1: GCA​AGU​ACU​UUG​UCG​ACU​UTT; siRNA-DmCLIC-2: CCA​CGU​UUA​CCA​CAU​ACA​UTT; siRNA-NC: UUC​UCC​GAA​CGU​GUC​ACG​UTT. Detailed information is listed in Supplementary Table S1.

For real-time quantitative PCR (RT‒qPCR) analysis, samples were collected at different time post-transfection. The related reaction was conducted with the CFX 96 Real-Time Detection System (Bio-Rad, CA, United States) using ChamQ SYBR qPCR Master Mix (without ROX) (Vazyme, Nanjing, China). Program conditions for thermal cycling were 95°C for 30 s, 40 cycles of 95°C for 5 s, and 60°C for 30 s. Three biological replicates were used for each treatment. Ct values were normalized to T. ni ribosomal protein S5, S. frugiperda actin, D. melanogaster ribosomal protein L32 and Homo sapiens GAPDH genes, respectively. The relative expression was calculated using the 2^-△△Ct method (Livak and Schmittgen, 2001). Primers were designed on Primer3web, and their sequences are listed in Supplementary Table S1.

Knockdown samples were collected and lysed with I-PER Insect Cell Protein Extraction Reagent (Thermo Fisher Scientific, MA, United States) at 48 h. The total protein concentration was quantified to the same amount before loading onto an 8%–16% SDS‒PAGE gel. For each sample, 4 × protein loading buffer was added, boiled (100°C, 10 min), and cooled in an ice bath for 10 min. Then, the separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Sigma, United States). The primary antibodies used were a rabbit anti-TnCLIC antibody and a mouse anti-β-actin antibody (diluted 1:2, 000). For the corresponding secondary antibodies, a goat anti-rabbit or goat anti-mouse IgG-horseradish peroxidase (HRP) conjugate was subsequently incubated (diluted 1:2, 000). The TnCLIC polyclonal antibody was prepared (HuaAn, Hangzhou, China) as above, and other antibodies were purchased commercially (GenScript, Nanjing, China). The chemiluminescence signal was developed using the Super Signal West Dura Extended Duration Substrate (Thermo Scientific, United States) and detected with a UVP Chemi Doc-It Imaging System (Upland, United States). Each experiment involved three biological replicates and the integrated density for each band was measured with Image J software (http://imagej.nih.gov/ij/, version 1.53).

Cell viability was measured using the RealTime-Glo™ MT Cell Viability Assay (Promega; Madison, WI, United States) according to the manufacturer’s instructions (Krall et al., 2021). The kit determines the number of viable cells in culture by measuring the reducing potential of cells and metabolism (MT); thus, the chemiluminescent signal correlates with the number of viable cells. In summary, the MT Cell Viability Substrate and NanoLuc® Enzyme were allowed to equilibrate to 37°C in a water bath before use. Then, both reagents were added to the cell suspension to a final concentration of 1X. After evenly mixing, cells were seeded on a 384-well assay plate at an equal density in a total volume of 70 μL per well and incubated at 27°C overnight. On the following day, 60%–80% confluent cells were transfected with siRNA at 100 nM final concentration as above. A Thermo Scientific Varioskan Flash (Thermo Scientific, Vantaa, Finland) was used to monitor luminescence at 48 h. Three replicates were performed for each treatment.

Cell death was evaluated by measuring the fluorescence intensity of the CellTox Green Cytotoxicity Assay (Promega; Madison, WI, United States) following the manufacturer’s protocol (Krzyzosiak et al., 2018). Fluorescence intensity correlates with the loss of cell membrane integrity caused by cell death. The final dilution of CellTox™ Green Dye (a proprietary asymmetric cyanine dye) to 1:1, 000 was provided, and for more detailed experimental procedures see the description above for the measurement of cell viability. Hi-5 cell morphology was observed using a Leica DMi8 (Leica, Wetzlar, Germany) after transfection at 96 h. To avoid the influence of the cell medium on the fluorescent value, the mix was removed before measuring fluorescence with a microplate reader at 485 nm excitation and 525 nm emission. Three independent replicates for each treatment condition were performed.

RNA extraction was conducted as described above. A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using the TruSeq RNA Library Preparation Kit (Illumina, United States), and index codes were added to attribute sequences to each sample. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Novaseq platform, and 150 bp paired-end reads were generated. To ensure the quality of the information analysis, the raw reads were filtered by fastp software (https://github.com/OpenGene/fastp). The main steps of data filtering were as follows: 1) remove reads with adaptors; 2) remove reads with an N content of more than 10%; and 3) remove reads with low-quality bases (quality value <20) accounting for more than 50% of the total bases. FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc) was then used for quality control of clean data. Transcripts were aligned to the reference Trichoplusia ni genome using HISAT2 (Kim et al., 2015). Gene expression levels were normalized by calculating fragments per kilobase of transcript per million mapped reads (FPKM), and a value of 0.1 was used as a threshold for expressed genes (high, FPKM ≥15; moderate, 3.75 ≤ FPKM <15; low, 0.1 ≤ FPKM <3.75). StringTie was used to estimate the expression levels of the detected genes (Pertea et al., 2015). Gene differential expression analysis was performed using DESeq2 software (Love et al., 2014). Genes with a false discovery rate (FDR) below 0.05 and a fold change ≥2 were considered differentially expressed genes (DEGs). DEGs were then subjected to enrichment analysis for GO functions and KEGG pathways with p-adjust (FDR) < 0.05 as the significance threshold using clusterProfiler software (https://github.com/GuangchuangYu/clusterProfiler). This work was performed by Wuhan GrandOmics Biotechnology Co., Ltd.

The calcium concentration was detected using a Calcium Colorimetric Assay kit (Beyotime, Shanghai, China) according to the manufacturer’s operating manual (Ni et al., 2021). Briefly, Hi-5 cells were collected and washed with PBS after siRNA transfection at 0, 12, 24, 48, and 96 h. The precooled lysate was added to the sample and fully lysed on a rotating platform at 4°C for 15 min. Then, the supernatant was carefully aspirated after centrifuging at 12, 000 × g for 5 min. A ready-to-use working solution (75 μL chromogenic reagent and 75 μL calcium assay buffer) was added to the 50 μL cell lysates, followed by a 10 min incubation at 37°C in the dark. Finally, a 96-well microplate reader was used to measure the absorbance at 575 nm. The standard curve for calcium content was developed according to the different gradients of the standard solution to calculate the calcium concentration of each sample.

Intracellular calcium ion levels were determinated using a Fluo-4 Calcium Assay Kit (Beyotime, Shanghai, China) according to the manual instructions (Huang et al., 2021). The cell culture and siRNA transfection were performed as described above. After each specified treatment time of incubation, Hi-5 cells were loaded with Fluo-4 AM dye and solubility enhancer (1:500) in assay buffer at 27°C in the dark for 30 min. Then, Fluo-4 staining solution was removed gently and corresponding fluorescence values (490 nm excitation, 525 nm emission) were recorded immediately using a microplate reader. The experiment was performed independently three times.

To chelate cytosolic Ca²⁺ signal transduction, Hi-5 cells were preloaded with BAPTA-AM (Aladdin, Shanghai, China) or EGTA-AM (AAT Bioquest, CA, United States) at 10 μM for 2 h in advance. After treatment, Hi-5 cells were transfected with siRNA as mentioned above. After 96 h, cell death was evaluated by CellTox™ Green Dye following the manufacturer’s protocol.

Intracellular mitochondrial membrane potential (MMP) signals were measured using a Mitochondrial Membrane Potential Assay Kit with Rhodamine 123 (Beyotime, Shanghai, China) (Qu et al., 2020), and reactive oxygen species (ROS) levels were assayed by a Reactive Oxygen Species Assay Kit (Beyotime, Shanghai, China) (Xin et al., 2022). In brief, Hi-5 cells were plated, maintained and transfected for 96 h. For the MMP assay, Hi-5 cells were washed with PBS after discarding the culture medium. Then, the cells were incubated with the working staining solution containing the cationic fluorescent probe 2-(6-amino-3-imino-3H-xanthen-9-yl) benzoic acid methyl ester (Rhodamine 123; 1:1, 000) at 37°C for 30 min. After discarding the supernatant, cell fluorescence was immediately detected at excitation (507 nm) and emission (529 nm) wavelengths. Hi-5 cells treated with 10 μM carbonyl cyanide 3-chlorophenylhydrazone (CCCP) for 20 min were used as a positive control. For intracellular ROS levels, Hi-5 cells were incubated with 2, 7-dichlorodihydrofluorescein diacetate (DCFH-DA; 1:1, 000) at 37°C for 20 min, which is easily oxidized to fluorescent dichlorofluorescein (DCF) by intracellular ROS. Hi-5 cells treated with a ROS-positive inducer (Rosup; 1:1, 000) for 30 min were used as a positive control. The fluorescence was detected at an excitation wavelength of 488 nm and at an emission wavelength of 525 nm after removing the supernatant.

Caspase 3/7 activation was measured using a Caspase-Glo® 3/7 Assay (Promega; Madison, WI, United States) according to the manufacturer’s protocol (Wang et al., 2017). Briefly, Hi-5 cells were grown in 96-well plates and treated with siRNA for 96 h. The ready-to-use reagent was prepared by swirling the buffer and substrate until the substrate was thoroughly dissolved. Afterward, an equal volume of reagent was added to each well and gently mixed. After incubation at room temperature for 30 min, the luminescence signal was immediately quantified using a microplate reader.

Hi-5 cells were seeded into a µ-Slide 4 Well (ibidi, Gräfelfing, Germany). The mitochondria and endoplasmic reticulums (ERs) in the cells were stained with 100 nM MitoTracker™ Red CMXRos (Thermo Fisher, CA, United States) and ER-Tracker Red (Beyotime, Shanghai, China) for 30 min, respectively. After washing with PBS quickly, the cells were fixed with 4% paraformaldehyde (PFA) at room temperature for 10 min. The samples were subsequently washed with PBS three times for 10 min each, permeabilized with 0.01% Triton X-100 for 15 min, blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature, and incubated with primary antibodies overnight at 4°C. The next day, after three washes with PBST (0.1% Tween-20) for 10 min each, the cells were incubated with secondary antibodies in the dark for 1 h at room temperature. After the samples were washed three times with PBST for 10 min each, the cell nuclei were stained with 1 μg/mL DAPI (GoodBiotech, Wuhan, China) for 10 min at room temperature. For the mitochondrial-associated membrane (MAM) marker, fatty acid CoA ligase 4 (FACL4) mouse monoclonal antibody (Beyotime, Shanghai, China) was used for the colocalization. Fluorescence signals were observed under a Zeiss LSM 880 confocal microscope (Carl Zeiss, Oberkochen, Germany).

Data were analyzed by Student’s t-test for comparison between two groups, and one-way ANOVA with Tukey’s multiple comparison test for comparison among multiple groups. Statistical calculations were performed using Data Processing System (DPS) software (Tang and Zhang, 2013). Figures were visualized by GraphPad Prism 5.0 software (GraphPad) and generated by AI Illustrator (Adobe).

The Trichoplusia ni chloride intracellular channel (TnCLIC) cDNA has a 783-bp ORF, encoding 260 amino acids, with a predicted molecular mass of 29.76 kDa and an isoelectric point of 5.66. Using the protein as a seed, we annotated CLIC in 110 insect species. The CLIC protein sequences selected for phylogenetic analysis are shown in the Supplementary material. Phylogenetic analysis results indicated that TnCLIC clustered with HzCLIC, HaCLIC, SfCLIC, and SlCLIC (Figure 1A). The corresponding species are Helicoverpa zea, H. armigera, S. frugiperda and S. litura. Among annotated genes, we found only one CLIC protein has been found in listed lepidopteran and coleopteran insects. No signal peptide was predicted for any insect CLIC protein sequence. Multiple sequence alignments (Figure 1B) showed that TnCLIC is similar to CLIC of other insect species from different taxonomic orders, but not Exc-4 or human CLIC4. It is implicated that CLIC is a unique and evolutionarily conserved protein in most insects.

To explore the function, we performed TnCLIC knockdown experiments in Hi-5 cells in vivo. The efficiency of siRNA silencing was assessed by RT‒qPCR using two different siRNA pairs (Figure 2A; Student’s t-test for 24 h and 48 h: t = 5.2718, df = 4, p = 0.0062; t = 3.7030, df = 4, p = 0.0208; t = 12.1935, df = 4, p = 0.0003; t = 4.0650, df = 4, p = 0.0153). The results showed that RNAi significantly downregulated TnCLIC expression at 24 and 48 h by siRNA-TnCLIC-1 compared with siRNA-TnCLIC-2. Thus, the protein level of TnCLIC knockdown at 48 h was evaluated using siRNA-TnCLIC-1 and the effect was validated by western blotting (Figures 2B, C; Student’s t-test for 48 h: t = 2.8087, df = 4, p = 0.0484). Correspondingly, luminescent and fluorescent values showed that siRNA-TnCLIC-1 inhibited cell proliferation at 48 h (Figure 2D; t = 3.7808, df = 6, p = 0.0092) and induced cell death in Hi-5 cells at 96 h (Figure 2E; t = 8.6923, df = 4, p = 0.0010) compared with the negative control. Bright and fluorescent images clearly proved that siRNA-TnCLIC-1 had lethal effects on the Hi-5 cell line at 96 h (Figure 2F). Meanwhile, Hi-5 cells transfected with siRNA-TnCLIC-1 displayed changes in the morphology, showing that treated cells formed significantly fewer colonies and changed from spindle-shaped to rounded and shrinkable. Together, these results indicate that TnCLIC is a protein essential for Hi-5 cell line survival.

To verify the lethal effects, CLIC knockdown experiments were performed in Sf9 and S2 cells in vivo. These siRNAs target SfCLIC or DmCLIC. Furthermore, these siRNAs were also respectively transfected into HEK 293T cells to verify siRNAs targeting insect CLIC genes had no significant effect on the human CLIC4 gene in HEK 293T cells. The interference efficiency was detected by RT‒qPCR at 48 h post-transfection using two pairs of siRNA (Figure 3A; Student’s t-test for 48 h: t = 10.1865, df = 4, p = 0.0005; t = 2.8983, df = 4, p = 0.0442; t = 72.5871, df = 4, p < 0.0001; t = 49.9480, df = 4, p < 0.0001; t = 0.4805, df = 4, p = 0.6560; t = 1.2606, df = 4, p = 0.2760). The results demonstrated the consequential outcome of reduced SfCLIC and DmCLIC expression in Sf9 and S2 cells, and siRNAs that target insect CLIC did not affect H. sapiens CLIC4 (HsCLIC4) expression since insect CLIC proteins are different from human CLIC4 by multiple sequence alignments. Besides a better interference efficiency, siRNA-1 targets similar sequence fragments for TnCLIC, SfCLIC and DmCLIC. Thus, siRNA-1 was used for follow-up experiments. Correspondingly, the protein level of CLIC knockdown was validated by western blotting (Supplementary Figure S2). In addition, luminescence and fluorescence signals showed that siRNA-1 inhibited cell proliferation (Figure 3B; t = 4.8206, df = 6, p = 0.0029; t = 6.9244, df = 6, p = 0.0004) and induced cell death at 48 h (Figure 3C; t = 4.3141, df = 4, p = 0.0125; t = 5.5414, df = 4, p = 0.0052) in Sf9 and S2 cells, rather than in the HEK 293T cell line (Figures 3B, C; ANOVA: F _{(2, 9)} = 1.8694, p = 0.2094; F _{(2, 6)} = 0.2356, p = 0.7971). It suggests that siRNAs that target insect CLIC only significantly induce cell death in insect cells but not in HEK 293T cells. This also indicates that siRNA-1 targeting SfCLIC or DmCLIC has no off-target effects on HEK 293T cells. In brief, these findings indicate that CLIC is vital for the survival of Sf9 and S2 insect cell lines.

To further identify the genes and pathways involved in the cell death phenotype of Hi-5 cells treated with siRNA-TnCLIC-1, the transcriptome profiles of siRNA-NC and siRNA-TnCLIC-1 lines were sequenced, compared and analyzed, referencing the Hi-5 cell line genome. Raw data for the transcriptome were deposited at NCBI sequence read archive (SRA) under BioProject PRJNA922752. One dataset of differentially expressed genes (DEGs) was generated as a result of the knockdown of TnCLIC: siRNA-NC vs. siRNA-TnCLIC-1. The DEGs affected by siRNA-TnCLIC-1 resulted in 1992 upregulated DEGs and 777 downregulated DEGs (Figure 4A). Then, Gene Ontology (GO: biological process, molecular function, and cellular component) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed. The results showed that the GO terms were significantly enriched in DNA binding, calcium ion binding and transmembrane transport (Figure 4B). The KEGG pathway analysis showed that the spliceosome, PI3K-Akt and calcium signaling pathways were significantly enriched for siRNA-NC vs. siRNA-TnCLIC-1 (Figure 4C). After GO and KEGG analyses, “calcium ion binding” and “calcium signaling” related terms and pathways were significantly co-enriched. To verify the relevant RNA-seq data, RT‒qPCR was performed and 11 candidate genes involved in Ca²⁺ signaling or encoding Ca²⁺ channels were significantly upregulated after TnCLIC knockdown (Figure 5; Student’s t-test in order: t = 18.2152, df = 4, p = 0.0001; t = 3.5751, df = 4, p = 0.0233; t = 4.1512, df = 4, p = 0.0142; t = 4.5661, df = 4, p = 0.0103; t = 4.4371, df = 4, p = 0.0114; t = 5.9185, df = 4, p = 0.0041; t = 7.2197, df = 4, p = 0.0020; t = 6.5403, df = 4, p = 0.0028; t = 8.0923, df = 4, p = 0.0013; t = 7.9005, df = 4, p = 0.0014; t = 7.9348, df = 4, p = 0.0014; t = 12.6456, df = 4, p = 0.0002). Thus, these results proved that TnCLIC widely participates in diverse pathways in response to knockdown, especially involving the levels of intracellular calcium ions.

To validate the results of transcriptomic analysis, the calcium concentration and cell death were detected using siRNA-TnCLIC-1 treatment for 0, 12, 24, 48, and 96 h against Hi-5 cells. Intracellular calcium concentration was detected by colorimetric and fluorescent methods, respectively. The results showed that the intracellular calcium concentration was significantly increased compared with that in the control (Figures 6A, B; Student’s t-test for 0 h: t = 1.6646, df = 4, p = 0.1713; t = 0.1652, df = 4, p = 0.8768. Student’s t-test for 12 h: t = 8.1095, df = 4, p = 0.0013; t = 19.9624, df = 4, p < 0.0001. Student’s t-test for 24 h: t = 27.2958, df = 4, p < 0.0001; t = 3.3588, df = 4, p = 0.0283. Student’s t-test for 48 h: t = 29.7562, df = 4, p < 0.0001; t = 21.8847, df = 4, p < 0.0001. Student’s t-test for 96 h: t = 307.9769, df = 4, p < 0.0001; t = 5.6127, df = 4, p = 0.0050). Correspondingly, cell death significantly increased (Figures 6A, B; Student’s t-test for 0 h: t = 0.1600, df = 4, p = 0.8806; t = 0.8375, df = 4, p = 0.4494. Student’s t-test for 12 h: t = 2.8804, df = 4, p = 0.0450; t = 3.5508, df = 4, p = 0.0238. Student’s t-test for 24 h: t = 3.2225, df = 4, p = 0.0322; t = 1.9195, df = 4, p = 0.1273. Student’s t-test for 48 h: t = 7.5309, df = 4, p = 0.0017; t = 5.2971, df = 4, p = 0.0061. Student’s t-test for 96 h: t = 4.9047, df = 4, p = 0.0080; t = 3.8863, df = 4, p = 0.0177). Then, correlation analyses of cell death and intracellular calcium ion were performed. Pearson and Spearman’s correlation analysis results show that intracellular calcium ion has a significant positive correlation with cell death (R = 0.8777, p < 0.0001; R = 0.9571, p < 0.0001). To further investigate the effect of Ca²⁺ in the cell death of TnCLIC mutant cells, Hi-5 cells were pretreated with Ca²⁺ chelators BAPTA-AM or EGTA-AM. After the treatment, cell death decreased significantly even after incubation with siRNA-TnCLIC-1 for 96 h (Figure 6C; ANOVA: F _{(3, 12)} = 61.1184, p < 0.0001; F _{(3, 12)} = 84.7996, p < 0.0001). In general, these findings indicate that cell death is involved in level increases of intracellular calcium ions after TnCLIC knockdown.

Various cellular signals, including the intracellular mitochondrial membrane potential (MMP), reactive oxygen species (ROS) and caspase 3/7 activity, were detected to uncover the cell death pattern. The results showed that intracellular MMP declined, and the ROS level and caspase 3/7 activity were increased when siRNA-TnCLIC-1 treatment was applied for 96 h (Figures 7A–C; ANOVA: F _{(2, 9)} = 107.8509, p < 0.0001; F _{(2, 9)} = 9.0822, p = 0.0069; Student’s t-test: t = 3.9841, df = 4, p = 0.0163). In addition, immunofluorescence images revealed that the TnCLIC protein located in the cytoplasm is mainly distributed in the mitochondria (Figure 7D) and partly distributed in the endoplasmic reticulum (Figure 7E; Supplementary Figure S4). Meanwhile, fatty acid CoA ligase 4 (FACL4), a marker enriched in the mitochondrial-associated membrane (MAM) fraction colocalizes with TnCLIC protein in Hi-5 cells.